Platelet rich plasma formulations and use thereof

ABSTRACT

Compositions for platelet rich plasma (PRP), depleted in neutrophils, are provided. Generally, these compositions comprise a higher concentration of platelets, lymphocytes and monocytes than whole blood with selective depletion of neutrophils to a concentration of less than about 5000/μl. These compositions may have depressed concentrations of red blood cells and hemoglobin. In some variations, the compositions may be useful to treat damaged connective tissue and/or to slow or stop cardiac apoptosis after a heart attack. The PRP composition may be delivered in conjunction with reperfusion therapy.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/333,082filed Dec. 21, 2011 which is a continuation of U.S. application Ser. No.12/576,101, filed Oct. 8, 2009 which claims priority to U.S. ProvisionalApplication No. 61/104,074, filed Oct. 9, 2008. All previousapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present application relates generally to formulations of a plateletrich plasma that may be used to treat various medical conditions. Morespecifically, the formulations of platelet rich plasma may comprisedifferent levels of platelets and white blood cells relative to wholeblood. In some variations, the formulations may comprise monocytes at aconcentration that is at least two times a monocyte concentration inwhole blood; lymphocytes at a concentration that is at least two times alymphocyte concentration in whole blood; and eosinophils at aconcentration that is about 1.5 times an eosinophil concentration inwhole blood.

BACKGROUND

Tissue damage and degeneration may be both a cause and an effect ofvarious medical conditions. Causes of damage may range from mechanicalinjury to other complex physiological processes involving inflammationand the like. For example, tissue damage may be the result of injury,overuse, reduced blood flow, or any other suitable cause. Even if thedamage is halted or slowed, the tissue may not completely heal due tothe formation of degenerative, immature, avascular, and scar tissue.

Connective tissues, such as tendons, ligaments, joint capsules, fascialtissues, and the like may be especially prone to damage. The overallprevalence of musculoskeletal disorders, for example, is approximately140 per 1000 persons in the United States, according to a 1995 survey bythe National Center for Health Statistics. The same survey estimated thedirect costs to be $88.7 billion and the indirect costs estimated to beup to $111.9 billion for lost productivity. Musculoskeletal injures maybe resistive to standard treatments such as anti-inflammatorymedication, bracing, rest and physical therapy. Injuries or other damageto flexible, relatively avascular connective tissues (hereafter“connective tissue” or “connective tissues”) may take a very long timeto heal (e.g., months or even years). In many cases, injuries toconnective tissues may never heal properly, and may require surgicalintervention.

One example of a musculoskeletal disorder is lateral epicondylitis.Lateral epicondylitis or “tennis elbow” is a well-known sports medicineand orthopedic disorder that is often associated with to overuse injuryand microtearing of the extensor carpi radialis brevis tendon at theelbow. The body attempts to repair these microtears but the healingprocess is incomplete in many cases. Pathologic specimens of patientsundergoing surgery for chronic lateral epicondylitis reveal adisorganized angiofibroblastic dysplasia. This incomplete attempt atrepair results in degenerative, immature, and avascular tissue. Thisincompletely repaired tissue may be weaker than normal tendon tissue andmay lack normal function. This inadequate healing may continue to causepain and may negatively impact the patient's ability to perform dailyactivities and the patient's quality of life.

Similar incomplete healing may be present in other types ofmusculoskeletal injuries or damage, such as patellar tendonitis(Jumper's Knee), Achilles tendonitis (common in runners), rotator cufftendonitis (commonly seen in “overhead” athletes such as baseballpitchers), chronic injuries of the ankle ligaments (“ankle sprains”), orligament tears.

Presently, many different non-operative and operative treatments exist.The non-operative measures include rest, activity modification, oralanti-inflammatory medication, and cortisone injections. While rest andactivity modification may help patients with some of these conditions,there remains a significant clinical population that is not adequatelytreated with these therapies. Despite widespread use, oralanti-inflammatory medications have not proven to be useful in controlledstudies. Some studies further suggest that non-steroidal medication mayactually have an adverse effect on the healing process for ligamentinjuries. Also, no acute inflammatory cells have been found inpathologic samples of cases of lateral epicondylitis. Cortisoneinjections are controversial in the treatment of tendinoses and arecontraindicated in acute ligament injuries. Several studies have notedan improvement in patients treated with cortisone in short term followup, but longer term results beyond one year have revealed a high symptomrecurrence rate and only an equivocal efficacy rate. These injectionsalso carry the risk of tendon rupture, infection, skin depigmentation,subdermal atrophy, and hyperglycemia in diabetic patients. The operativemeasures include debridement and repair of the associated pathologictendons. However, open or arthroscopic surgery has many potentialcomplications such as deep infection, damage to neurovascularstructures, and scar formation. The surgery is also expensive andcarries the additional risks associated with regional or generalanesthesia.

While musculoskeletal injuries may be associated with physical ormechanical processes, other types of tissue injury may involvephysiological processes. For example, myocardial injury from acompromised cardiac vascular system may result in cell ischemia or evencell death. According to the American Heart Association, coronary heartdisease is the single leading cause of death in the United States. Theprevalence of heart attack in the U.S. was approximately 8.1 millionpeople in 2005, and, of those, 920,000 were new or recurrent. Heartattack is also known as acute myocardial infarction (MI) and occurs whenthe blood supply to the heart is interrupted—usually by a plaquedetaching from and blocking a cardiac blood vessel. As a result ofrestricted blood flow, the adjacent cardiac tissue becomes ischemicbegins to die. If left untreated, an MI will lead to death.

Acute myocardial infarction may comprise non-ST-elevated myocardialinfarction or ST-elevated myocardial infarction. In an ST-elevatedmyocardial infarction, the ST segment in an electrocardiogram (ECG) iselevated, meaning that the ventricles do not depolarize as rapidly asthey would in a healthy heart. If blood flow to the heart is impairedover an extended period of time, an ischemic cascade and cardiacapoptosis may occur, causing heart cells to die and not regenerate. Inplace of the ischemic tissue, scar tissue forms. The scar tissue mayincrease the likelihood of cardiac arrhythmia, and may result in theformation of ventricular aneurysms.

To treat an MI, reperfusion therapy may be performed. Reperfusiontherapies include thrombolytic therapy, percutaneous coronaryintervention (PCI), and/or bypass surgery. While reperfusion therapyrestores blood flow to the ischemic tissue, it does not lessen the riskof arrhythmia resulting from the growth of scar tissue. Because of theheightened risk of arrhythmia, the patient may be placed onanti-arrhythmia agents and/or require a pacemaker.

As such, additional treatments for treating tissue damage are desirable.Kits for treating tissue damage are also desirable.

SUMMARY

Platelet rich plasma (PRP) compositions for treating tissue damage areprovided. The compositions may generally comprise a platelet rich plasmathat includes a specific concentration of platelets, red blood cells,and white blood cells. In some examples, the compositions may becharacterized relative to a baseline concentration of the platelets, redblood cells, and/or white blood cells of the whole blood from which thecompositions are directly or indirectly derived. The PRP compositiondisclosed herein may be useful in treating connective tissue and/orcardiac tissue. For example, it is believed that the PRP compositionsdescribed herein may repair tissue damage by slowing or haltingapoptosis, and that the anti-apoptotic effects of the PRP compositionsmay be measured based on a decrease in caspases in the blood, such ascaspase-3.

In some examples, the PRP composition comprises platelet cells at aconcentration at least 1.1 times the concentration of platelets in wholeblood. The platelet concentration in the PRP composition may be betweenabout 1.1 and about 2 times baseline, about 2 and about 4 timesbaseline, about 4 and about 6 times baseline, about 6 and about 8 timesbaseline, or higher. The platelet concentration in the PRP compositionmay be between about 500,000 and about 1,500,000 platelets permicroliter.

The PRP composition may further comprise white blood cells (WBCs) at ahigher concentration than white blood cells in whole blood. The WBCconcentration may be between about 1.1 and about 2 times baseline, about2 and about 4 times baseline, about 4 and about 6 times baseline, about6 and about 8 times baseline, or higher. In some variations, the WBCconcentration is about 15,000 to about 50,000 WBC per microliter.

In some variations, the PRP composition may comprise specificconcentrations of various types of white blood cells. The concentrationsof lymphocytes and monocytes may be between about 1.1 and about 2 timesbaseline, about 2 and about 4 times baseline, about 4 and about 6 timesbaseline, about 6 and about 8 times baseline, or higher. Theconcentrations of eosinophils in the PRP composition may be about 1.5times baseline. In some variations, the lymphocyte concentration isbetween about 5,000 and about 20,000 per microliter and the monocyteconcentration is between about 1,000 and about 5,000 per microliter. Theeosinophil may be between about 200 and about 1,000 per microliter.

In certain variations, the PRP composition may contain a specificconcentration of neutrophils. The neutrophil concentration may varybetween less than the baseline concentration of neutrophils to eighttimes the baseline concentration of neutrophils. In some variations, theneutrophil concentration may be between 0 and about 0.1 times baseline,about 0.1 and about 0.5 times baseline, about 0.5 and 1.0 timesbaseline, about 1.0 and about 2 times baseline, about 2 and about 4times baseline, about 4 and about 6 times baseline, about 6 and about 8times baseline, or higher. The neutrophil concentration may additionallyor alternatively be specified relative to the concentration of thelymphocytes and/or the monocytes. In preferred embodiments, theneutrophil concentration is less than the concentration in whole blood.In a more preferred embodiment, the neutrophil concentration is 0.1 to0.9 the concentration found in whole blood, yet more preferably lessthan 0.1 the concentration found in whole blood. In a most preferredembodiment the neutrophils are eliminated or non-detectable in the PRPcomposition.

In some embodiments, the PRP compositions may comprise a lowerconcentration of red blood cells (RBCs) and/or hemoglobin than theconcentration in whole blood. The RBC concentration may be between about0.01 and about 0.1 times baseline, about 0.1 and about 0.25 timesbaseline, about 0.25 and about 0.5 times baseline, or about 0.5 andabout 0.9 times baseline. The hemoglobin concentration may be 5 g/dl orless.

The PRP compositions are typically generated from whole blood orportions of whole blood using a variety of techniques comprising, forexample, centrifugation, gravity filtration, and/or direct cell sorting.Once generated the PRP compositions may undergo one or more processes toconfirm the concentrations and/or activation of the various components.

In preferred embodiments, any of the PRP compositions described aboveare prepared from a patient who has not been previously treated with athrombolytic agent, such as heparin, tPA, PLAVIX®, or aspirin.Preferably, the patient has not received a thrombolytic agent for atleast 2 hours, preferably 1 day, more preferably 2 weeks, and yet morepreferably 1 month prior to withdrawing the blood for extraction of thePRP. In particular, if a patient is a candidate for reperfusion therapy,the blood to be used for extraction of PRP is drawn from the patientbefore administering the reperfusion therapy.

Embodiments of the invention are directed to methods of identifying drugcandidates for treating a disease or condition based upon a response toa PRP composition, such as a PRP composition described above. Inpreferred embodiments, a PRP composition as described above isadministered as a treatment to an individual suffering from a disease orcondition. Alternatively, a model for the disease could be used such asan animal or cell culture system. The PRP composition is administered tothe model animal or included in the cell culture media. In otherembodiments, simulations are carried out using a computer. The efficacyof the treatment is monitored in the individual or animal model or inthe cell culture or the computer simulation. Individuals responsive tothe treatment are selected and a sample is obtained from the responsiveindividual. In the case of a human patient or animal model, this samplemight be a bodily fluid sample such as blood or saliva or a tissuesample. In a cell culture, the responsive cells are selected. In acomputer model, positive simulations are identified.

Analysis is performed on the biological sample obtained from thepatient, animal or cell cultures. Typically such analysis would be by animmunoassay to determine the presence of specific antibodies or antigensor a genetic analysis. The genetic analysis may indicate genes that areupregulated or downregulated. In the case of a computer simulation,parameters are identified indicative of a positive response.

The results obtained as above are compared to results obtained from anon-disease population or a subpopulation having the disease but notresponsive to treatment to determine targets present in the responsivepopulation. Based upon the identified targets, drugs candidates, such asproteins or small molecules, for treating the disease condition areidentified and further tested.

In preferred embodiments, the disease or condition is ischemia, cancer,a disease of the immune system, a connective tissue injury, a skindisease, or a disease of the nervous system. The ischemia may be a brainischemia or cardiac ischemia. The cancer may be brain cancer, thyroidcancer, pancreatic cancer, liver cancer, breast cancer, or prostatecancer. The connective tissue injury may be a tendinosis, such as tenniselbow, rotator cuff injury, a knee injury, a spinal injury or plantarfasciitis. The nervous system disease may be Parkinsons' disease.

In preferred embodiments, the PRP composition is depleted inneutrophils, preferably at a level of 0 to 0.9 of the concentration ofwhole blood.

In preferred embodiments, the analysis involves generation of a geneticprofile. For example a DNA array may be used to determine defectivegenes or may be used to determine patterns of gene expression. In somepreferred embodiments, the analysis involves measuring levels ofproteins, lipids, antibodies, antigens, enzymes or small molecules.

Embodiments of the invention are directed to a method of diseasediagnosis for a patient by one or more of the following steps:

obtaining a blood sample from the patient;

obtaining PRP from the blood sample;

obtaining an analysis from the PRP; and

comparing the analysis of the PRP from the patient with an analysisobtained from PRP of a population having the disease; and

determining that analysis of the PRP of the patient matches the analysisof the PRP of the disease population, thereby diagnosing the disease. Inpreferred embodiments, the analysis is an immunoassay or geneticprofile.

Embodiments of the invention are directed to a device having plateletrich plasma composition as described above alone or in combination witha fixation device such as a stent, suture, screw or implantable device.Preferably, the device is a chamber. More preferably, conditions in thechamber include one or more of the following: low oxygen tension, highoxygen tension, low pH, high pH, low pressure, high pressure, low UV,high UV, low temperature and high temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table of an exemplary PRP composition used in murine models.

FIG. 2 is a graph comparing the cardiac ejection fraction of subjectstreated with the composition to a control group.

DETAILED DESCRIPTION Overview

Biological compositions that can be used to treat various medicalconditions are provided. The biological compositions generally comprisea platelet rich plasma that includes white blood cells and platelets athigher concentrations than those in whole blood. In connective tissue,this PRP composition may be used to treat tissue that has been damageddue to injury, wound, trauma, lesion, and/or tissue degeneration. Incardiac tissue, this PRP composition may be used to treat ischemictissue following an acute MI to preserve myocardial tissue and promotere-growth. Additionally, the PRP composition may slow or halt apoptosis,reduce infarct size, decrease cardiac arrhythmias, and/or restore cellfunction.

It has been hypothesized that the PRP composition may promote regrowthby slowing or halting apoptosis. For example, in cardiac tissue, cardiacapoptosis is a process in metazoan cells that contributes to thedevelopment of congestive heart failure. Cardiac apoptosis may reducethe number of contractile cardiocytes in the myocardium throughprogrammed cell death. The low, but abnormal, rate of cardiocyteapoptosis may be a significant loss for adult human myocardium, at leastin part because adult human cardiocytes have typically lost the abilityto replicate. Apoptosis of cardiac non-myocytes may diminish thecontractile mass, which may also lead to heart failure. Apoptosis ofcardiac non-myocytes may also contribute to maladaptive remodeling andthe subsequent transition to decompensated congestive heart failure.

Cardiac apoptosis may be measured by the level of cysteine proteases inthe blood. Specifically, caspase-3 is a cysteine-aspartic acid proteasethat may be used as a marker for cardiac apoptosis. Generally, caspase-3is an effector caspase that cleaves to protein substrates within thecell resulting in the apoptotic process. A caspase cascade may occurwhen the caspase-3 is automatically activated between cells. In cardiactissue, caspase-3 has been shown to significantly rise as early as oneday after a myocardial infarction and persist for up to four weeks.

To treat tissue damaged by, for example, apoptosis, injury, wound,trauma, lesion, or degeneration, various methods for delivering the PRPcomposition into the connective tissue and/or the myocardium aredisclosed. In various embodiments, the composition may be delivered todamaged connective tissues, the region of connective tissue directlyadjacent to the damaged tissue, and/or healthy tissue. In someembodiments, the PRP composition may be delivered to the ischemictissues, the region of tissue directly adjacent to the ischemic tissue,and/or healthy tissue. The PRP composition may comprise a platelet gel,or flowable material or liquid, other substances described herein, orany substance suitable for providing the desired level of treatment ofthe damaged or ischemic tissues.

The PRP composition may be delivered to a patient in an emergencysituation or as part of an elective procedure. To treat damagedconnective tissue, the PRP composition may be delivered as part of aninpatient or outpatient procedure days, weeks, months, or years afterthe tissue damage occurred. Examples of connective tissue damage thatmay be treated using PRP include, but are not limited to, lateralepicondylitis (i.e., tennis elbow), plantar fasciitis, patellartendonitis (i.e., Jumper's Knee), Achilles tendonitis, rotator cufftendonitis, ankle sprains, and ligament tears. The tissue damage may beidentified using one or more medical imaging technologies such as, butnot limited to, x-ray imaging, magnetic resonance imaging (MRI), andultrasound imaging. To treat damage to the myocardium, the PRPcomposition may be delivered in an emergency room and/or by emergencymedical service providers when an MI is identified. In other instances,the PRP composition may be delivered after an MI during reperfusiontherapy.

The MI may be identified by determining whether enzymes such as cardiactroponin (e.g., troponin-I or T), creatine kinase (CK) including CK-MB,aspartate transminase (AST)/Glutamic Oxaloacetic Transaminase(GOT/SGOT)/aspartate aminotransferase (ASAT), lactate dehydrogenase(LDH), and/or myoglobin (Mb), and/or the like are present in the bloodstream. The PRP compositions described herein may be delivered in theabsence of the enzymes. Myocardial infarctions may be determined byidentifying ST elevation in an ECG (e.g., during rest, a pharmacologicalstress test, and/or a physiological stress test), by coronary angiogram(e.g., noting acute closure of a vessel supplying myocardium at risk),by a nuclear medicine scan (e.g., technetium-99m or thalium-201), etc.

The PRP compositions used to treat the damaged connective tissue or themyocardium may be generated from whole blood or portions of whole bloodusing a variety of techniques comprising, for example, centrifugation,gravity filtration, and/or direct cell sorting. Once generated, the PRPcompositions may undergo one or more processes to confirm theconcentrations and/or activation of the various components.

The compositions, devices, methods, and kits described herein areillustrative of various embodiments, variations, and adaptations. Thedisclosure is not intended to be limited to only the embodimentsdescribed.

Compositions

The PRP compositions generally comprise elevated concentrations ofplatelets and WBCs relative to whole blood. Typically, the concentrationof RBCs and hemoglobin is depressed. In some variations, theconcentrations of the platelets and the WBCs are specified to increasethe likelihood that the PRP composition will effectively treat damagedtissue and/or ischemic tissue.

The PRP composition generally includes platelets at a plateletconcentration that is higher than the baseline concentration of theplatelets in whole blood. Baseline concentration means the concentrationfound in the patient's blood which would be the same as theconcentration found in a blood sample from that patient withoutmanipulation of the sample by laboratory technique such as cell sorting,centrifugation or filtration. The platelet concentration may be betweenabout 1.1 and about 2 times the baseline, about 2 and about 3 times thebaseline, about 3 and about 4 times the baseline, about 4 and about 5times the baseline, about 5 and about 6 times the baseline, about 6 andabout 7 times the baseline, about 7 and about 8 times the baseline,about 8 and about 9 times the baseline, about 9 and about 10 times thebaseline, about 11 and about 12 times the baseline, about 12 and about13 times the baseline, about 13 and about 14 times the baseline, orhigher. In some embodiments, the platelet concentration may be betweenabout 4 and about 6 times the baseline. Typically, a microliter of wholeblood comprises at least 140,000 to 150,000 platelets and up to 400,000to 500,000 platelets. The PRP compositions may comprise about 500,000 toabout 7,000,000 platelets per microliter. In some instances, the PRPcompositions may comprise about 500,000 to about 700,000, about 700,000to about 900,000, about 900,000 to about 1,000,000, about 1,000,000 toabout 1,250,000, about 1,250,000 to about 1,500,000, about 1,500,000 toabout 2,500,000, bout 2,500,000 to about 5,000,000, or about 5,000,000to about 7,000,000 platelets per microliter.

The WBC concentration is typically elevated in the PRP composition. Forexample, the WBC concentration may be between about 1.1 and about 2times the baseline, about 2 and about 4 times the baseline, about 4 andabout 6 times the baseline, about 6 and about 8 times the baseline,about 8 and about 10 times the baseline, or higher. The WBC count in amicroliter of whole blood is typically at least 4,100 to 4,500 and up to10,900 to 11,000. The WBC count in a microliter of the PRP compositionmay be between about 8,000 and about 10,000, about 10,000 and about15,000, about 15,000 and about 20,000, about 20,000 and about 30,000,about 30,000 and about 50,000, about 50,000 and about 75,000 and about75,000 and about 100,000.

Among the WBCs in the PRP composition, the concentrations may vary bythe cell type but, generally, each may be elevated. In some variations,the PRP composition may comprise specific concentrations of varioustypes of white blood cells. The relative concentrations of one cell typeto another cell type in a PRP composition may be the same or differentthan the relative concentration of the cell types in whole blood. Forexample, the concentrations of lymphocytes and/or monocytes may bebetween about 1.1 and about 2 times baseline, about 2 and about 4 timesbaseline, about 4 and about 6 times baseline, about 6 and about 8 timesbaseline, or higher. In some variations, the concentrations of thelymphocytes and/or the monocytes may be less than the baselineconcentration. The concentrations of eosinophils in the PRP compositionmay be less than baseline, about 1.5 times baseline, about 2 timesbaseline, about 3 times baseline, about 5 times baseline, or higher.

In whole blood, the lymphocyte count is typically between 1,300 and4,000 cells per microliter, but in other examples, the lymphocyteconcentration may be between about 5,000 and about 20,000 permicroliter. In some instances, the lymphocyte concentration may be lessthan 5,000 per microliter or greater than 20,000 per microliter. Themonocyte count in a microliter of whole blood is typically between 200and 800. In the PRP composition, the monocyte concentration may be lessthan about 1,000 per microliter, between about 1,000 and about 5,000 permicroliter, or greater than about 5,000 per microliter. The eosinophilconcentration may be between about 200 and about 1,000 per microliterelevated from about 40 to 400 in whole blood. In some variations, theeosinophil concentration may be less than about 200 per microliter orgreater than about 1,000 per microliter.

In certain variations, the PRP composition may contain a specificconcentration of neutrophils. The neutrophil concentration may varybetween less than the baseline concentration of neutrophils to eighttimes than the baseline concentration of neutrophils. In somevariations, the neutrophil concentration may be between about 0.01 andabout 0.1 times baseline, about 0.1 and about 0.5 times baseline, about0.5 and 1.0 times baseline, about 1.0 and about 2 times baseline, about2 and about 4 times baseline, about 4 and about 6 times baseline, about6 and about 8 times baseline, or higher. The neutrophil concentrationmay additionally or alternatively be specified relative to theconcentration of the lymphocytes and/or the monocytes. One microliter ofwhole blood typically comprises 2,000 to 7,500 neutrophils. In somevariations, the PRP composition may comprise neutrophils at aconcentration of less than about 1,000 per microliter, about 1,000 toabout 5,000 per microliter, about 5,000 to about 20,000 per microliter,about 20,000 to about 40,000 per microliter, or about 40,000 to about60,000 per microliter. In preferred embodiments, neutrophils areeliminated or substantially eliminated. Means to deplete blood products,such as PRP, of neutrophils is known as discussed in U.S. Pat. No.7,462,268, which is incorporated herein by reference.

Typically, whole blood drawn from a male patient may have an RBC countof at least 4,300,000 to 4,500,000 and up to 5,900,000 to 6,200,000 permicroliter while whole blood from a female patient may have an RBC countof at least 3,500,000 to 3,800,000 and up to 5,500,000 to 5,800,000 permicroliter. These RBC counts generally correspond to hemoglobin levelsof at least 132 g/L to 135 g/L and up to 162 g/L to 175 g/L for men andat least 115 g/L to 120 g/L and up to 152 g/L to 160 g/L for women.

In some embodiments, the PRP compositions may comprise a lowerconcentration of red blood cells (RBCs) and/or hemoglobin than theconcentration in whole blood. The RBC concentration may be between about0.01 and about 0.1 times baseline, about 0.1 and about 0.25 timesbaseline, about 0.25 and about 0.5 times baseline, or about 0.5 andabout 0.9 times baseline. The hemoglobin concentration may be depressedand in some variations may be about 1 g/dl or less, between about 1 g/dland about 5 g/dl, about 5 g/dl and about 10 g/dl, about 10 g/dl andabout 15 g/dl, or about 15 g/dl and about 20 g/dl.

Methods of Making

The PRP composition may comprise a PRP derived from a human or animalsource of whole blood. The PRP may be prepared from an autologoussource, an allogenic source, a single source, or a pooled source ofplatelets and/or plasma. To derive the PRP, whole blood may becollected, for example, using a blood collection syringe. The amount ofblood collected may depend on a number of factors, including, forexample, the amount of PRP desired, the health of the patient, theseverity or location of the connective tissue damage and/or the MI, theavailability of prepared PRP, or any suitable combination of factors.Any suitable amount of blood may be collected. For example, about 20 ccto about 150 cc of blood may be drawn. More specifically, about 27 cc toabout 110 cc or about 27 cc to about 55 cc of blood may be withdrawn. Insome embodiments, the blood may be collected from a patient who may bepresently suffering, or who has previously suffered from, connectivetissue damage and/or an MI. PRP made from a patient's own blood maysignificantly reduce the risk of adverse reactions or infection.

In an exemplary embodiment, about 55 cc of blood may be withdrawn into a60 cc syringe (or another suitable syringe) that contains about 5 cc ofan anticoagulant, such as a citrate dextrose solution. The syringe maybe attached to an apheresis needle, and primed with the anticoagulant.Blood (about 27 cc to about 55 cc) may be drawn from the patient usingstandard aseptic practice. In some embodiments, a local anesthetic suchas anbesol, benzocaine, lidocaine, procaine, bupivicaine, or anyappropriate anesthetic known in the art may be used to anesthetize theinsertion area.

The PRP may be prepared in any suitable way. For example, the PRP may beprepared from whole blood using a centrifuge. The whole blood may or maynot be cooled after being collected. Isolation of platelets from wholeblood depends upon the density difference between platelets and redblood cells. The platelets and white blood cells are concentrated in thelayer (i.e., the “buffy coat”) between the platelet depleted plasma (toplayer) and red blood cells (bottom layer). For example, a bottom buoyand a top buoy may be used to trap the platelet-rich layer between theupper and lower phase. This platelet-rich layer may then be withdrawnusing a syringe or pipette. Generally, at least 60% or at least 80% ofthe available platelets within the blood sample can be captured. Theseplatelets may be resuspended in a volume that may be about 3% to about20% or about 5% to about 10% of the sample volume.

In some examples, the blood may then be centrifuged using agravitational platelet system, such as the Cell Factor Technologies GPSSystem® centrifuge. The blood-filled syringe containing between about 20cc to about 150 cc of blood (e.g., about 55 cc of blood) and about 5 cccitrate dextrose may be slowly transferred to a disposable separationtube which may be loaded into a port on the GPS centrifuge. The samplemay be capped and placed into the centrifuge. The centrifuge may becounterbalanced with about 60 cc sterile saline, placed into theopposite side of the centrifuge. Alternatively, if two samples areprepared, two GPS disposable tubes may be filled with equal amounts ofblood and citrate dextrose. The samples may then be spun to separateplatelets from blood and plasma. The samples may be spun at about 2000rpm to about 5000 rpm for about 5 minutes to about 30 minutes. Forexample, centrifugation may be performed at 3200 rpm for extraction froma side of the separation tube and then isolated platelets may besuspended in about 3 cc to about 5 cc of plasma by agitation. The PRPmay then be extracted from a side port using, for example, a 10 ccsyringe. If about 55 cc of blood may be collected from a patient, about5 cc of PRP may be obtained.

As the PRP composition comprises activated platelets, active agentswithin the platelets are released. These agents include, but are notlimited to, cytokines (e.g., IL-1B, 1L-6, TNF-A), chemokines (e.g.,ENA-78 (CXCL5), IL-8 (CXCL8), MCP-3 (CCL7), MIP-1A (CCL3), NAP-2(CXCL7), PF4 (CXCL4), RANTES (CCL5)), inflammatory mediators (e.g.,PGE2), and growth factors (e.g., Angiopoitin-1, bFGF, EGF, FGF, HGF,IGF-I, IGF-II, PDAF, PDEGF, PDGF AA and BB, TGF-.beta. 1, 2, and 3, andVEGF).

The PRP composition may be delivered as a liquid, a solid, a semi-solid(e.g., a gel), an inhalable powder, or some combination thereof. Whenthe PRP is delivered as a liquid, it may comprise a solution, anemulsion, a suspension, etc. A PRP semi-solid or gel may be prepared byadding a clotting agent (e.g., thrombin) to the PRP. The gel may be moreviscous than a solution and therefore may better preserve its positiononce it is delivered to target tissue.

In some instances, it may be desirable to deliver the PRP composition asa liquid and have it gel or harden in situ. For example, the PRPcompositions may include, for example, collagen, cyanoacrylate,adhesives that cure upon injection into tissue, liquids that solidify orgel after injection into tissue, suture material, agar, gelatin,light-activated dental composite, other dental composites, silk-elastinpolymers, Matrigel® gelatinous protein mixture (BD Biosciences),hydrogels and/or other suitable biopolymers. Alternatively, the abovementioned agents need not form part of the PRP mixture. For example, theabove mentioned agents may be delivered to the target tissue before orafter the PRP has been delivered to the target tissue to cause the PRPto gel. In some embodiments, the PRP composition may harden or gel inresponse to one or more environmental or chemical factors such astemperature, pH, proteins, etc.

The PRP may be buffered using an alkaline buffering agent to aphysiological pH. The buffering agent may be a biocompatible buffer suchas HEPES, TRIS, monobasic phosphate, monobasic bicarbonate, or anysuitable combination thereof that may be capable of adjusting the PRP tophysiological pH between about 6.5 and about 8.0. In certainembodiments, the physiological pH may be from about 7.3 to about 7.5,and may be about 7.4. For example, the buffering agent may be an 8.4%sodium bicarbonate solution. In these embodiments, for each cc of PRPisolated from whole blood, 0.05 cc of 8.4% sodium bicarbonate may beadded. In some embodiments, the syringe may be gently shaken to mix thePRP and bicarbonate.

As noted above, the PRP composition may comprise one or more additionalagents, diluents, solvents, or other ingredients. Examples of theadditional agents include, but are not limited to, thrombin,epinephrine, collagen, calcium salts, pH adjusting agents, materials topromote degranulation or preserve platelets, additional growth factorsor growth factor inhibitors, NSAIDS, steroids, anti-infective agents,and mixtures and combinations of the foregoing.

Furthermore, the PRP compositions may comprise a contrast agent fordetection by an imaging technique such as X-rays, magnetic resonanceimaging (MRI), or ultrasound. Examples of such contrast agents include,but are not limited to, X-ray contrast (e.g., IsoVue), MRI contrast(e.g., gadolinium), and ultrasound contrast.

Methods of Testing

In some variations, the PRP composition may be analyzed and/or modifiedprior to delivery to the patient. The PRP composition may be modifiedbased on, for example, the condition to be treated, an initial completeblood count, a genetic profile of the patient, and other suitablefactors.

In some embodiments, a patient's genetic profile is determined. The PRPcomposition of healthy individuals having the same or similar geneticprofile is determined. A PRP composition is prepared in which componentsare matched to the PRP of the healthy individual having the same geneticprofile. The modified PRP composition is administered to the patient totreat the disease or condition.

In some embodiments, the PRP composition of a patient, successfullyrecovering from a disease or condition may be used as a model to preparea PRP composition to administer to a patient diagnosed with the samedisease or condition. In other words, the PRP composition is firstenriched in components which are effective in treating the disease basedupon recovered or recovering individuals. The modified PRP compositionis then administered to the patient suffering from the disease.

The PRP, or a portion of the PRP, may be placed into an automated bloodanalyzer that performs a compete blood count (CBC). As part of the CBC,the automated blood analyzers typically return a count of the number ofplatelets, WBCs, and RBCs present in the sample. The WBC count mayfurther include counts of lymphocytes, monocytes, basophils,neutrophils, and/or eosinophils. Examples of blood analyzers that may beused include, but are not limited to, Beckman Coulter LH series, SysmexXE-2100, Siemens ADVIA 120 & 2120, and the Abbott Cell-Dyn series.

It is believed that the effectiveness of treatments using PRP may be atleast partially dependent on the genetic profile of the patient. Thewhole blood of a patient may be tested before and/or after generatingthe PRP composition to determine if the PRP composition is likely toaffect the ability of the tissue to regenerate. Once the PRP has beendetermined to be useful, it may be delivered to the patient.

In certain variations, one or more genetic markers of a patient's DNA,mRNA, proteins, or the like may be evaluated prior to, during, and/orafter delivery of the PRP composition. The patient's DNA, or otherbiomarkers, is typically captured via a sample such as blood, saliva, orother suitable body fluid or body tissue. The sample may be tested forgenetic markers that are correlatable to the effectiveness of treatmentsusing the PRP composition. In some instances, the identified geneticmarkers may be detectable using a genetic tool such as a gene chip orother genetic expression technology. In some instances, the genes thatmay be tested for include, but are not limited to, collagen type I(COL1A1), collagen type III (COL3A1), cartilage oligomeric matrixprotein (COMP), matrix metalloproteinase-3 (MMP-3), and matrixmetalloproteinase-13 (MMP-13). Such genetic tools can be used to measurechanges in expression levels, or to detect single nucleotidepolymorphisms (SNPs) which may be associated with a disease condition.Many gene chips are commercially available including the Affymetrix GeneChip®, the Applied Microarrays CodeLink® arrays, and the EppendorfDualChip & Silverquant®.

In some variations, the genetic tool may be analyzed to determine if thepatient is likely to respond (favorably or unfavorably) to the PRPcomposition and/or to subsequent treatments. In certain variations, thePRP composition may be tested at a range of pH values and/or the pH ofthe PRP may be modified based at least in part on the genetic profile.In some instances, various genetic profiles may be associated withspecific concentrations (or ranges of concentrations) as being more orless effective than other concentrations for various components of thePRP composition. The response to the PRP composition may be slowing orhalting of cardiac apoptosis, anti-arrhythmia effects, or otherwisedecrease risks associated with reperfusion therapy.

If the CBC returned by the automated blood analyzer is not withinspecified ranges, the PRP composition may be modified using a filtrationdevice and/or cell sorter. The filtration device may use vacuum and/orgravity to remove a portion of the platelet, WBCs, and/or RBCs. In somevariations, a cell sorter may receive a CBC input from an automatedblood analyzer and/or a gene chip reader. A user may select or confirmone or more modifications to be made to the PRP composition. Of course,the cell sorter may be used with whole blood, portions of whole blood,and/or PRP. The cell sorter may sort the PRP composition based onelectric charge, density, size, deformation, fluorescence, or the like.Examples of cell sorters include the BD FACSAria® cell sorter, theCytopeia InFlux® cell sorter, those manufactured by Beckman Coulter, theCytonome Gigasort® cell sorter, and the like.

Use of Platelet Rich Plasma Compositions in Drug Discovery

Embodiments of the invention are directed to the use of platelet richplasma compositions as described herein in drug discovery. A PRPcomposition is administered in a model system, preferably a mammalianmodel, such as a disease model or in the course of a human study or in anon-mammalian system such as a plant. The effects of the administeredPRP composition on gene expression is monitored. For example, in oneembodiment, the effect of a PRP composition in a cell culture system isstudied by molecular analysis of the cells. DNA, RNA, microRNA, and/orepigenetic markers are evaluated to determine efficacy of drugs for aspecific disorder. In some preferred embodiments, the mechanism by whichplatelet rich plasma prevents apoptosis in ischemic tissue or cells isstudied to discover an existing or new protein that acts in a ratelimiting step or critical path. This protein is then purified and usedas a small molecule drug in the treatment of a disease. Dosage of drugsis also evaluated.

In a preferred embodiment, a PRP composition is used in a disease model(in-vitro, animal, human, computer) to evaluate gene expression.Preferably, the disease model is a cell or tissue culture or an animalmodel or a human study. The PRP composition is used as a test treatmentin the disease model compared to no treatment or other known treatments.DNA microarray, RNA, microRNA, epigenetics or other molecular analysistechniques are used to determine changes in gene expression due to thePRP treatment in the model. Cellular gene expression is evaluated andanalyzed for patterns. Identified molecules are purified. Drugs aregenerated for treatment of the specific disorder and tested forefficacy. Specifically, enzymes, proteins or molecules that may be usedto treat a specific disorder or condition are identified.

In some embodiments, drug screening is specific for a patient orsubpopulation of patients having a disease or condition. In someembodiments, a platelet rich plasma composition is administered to apatient suffering from a disease or condition and the effectiveness ofthe PRP composition is monitored in the patient. If the treatment iseffective, a sample is taken from the patient, typically a bodily fluidsuch as blood or saliva or a tissue sample. The sample is analyzed formarkers associated with recovery. The sample is used to determine thegenetic profile of the patient using a DNA array (gene chip) or specificmarkers. This profile is compared to patients not responsive to thetreatment. These may be diseased individual that did not respond totreatment with the PRP composition or healthy individuals. Based uponthe difference in genetic profile, specific genes are identified as drugtargets for the patient population responsive to the PRP composition.Other markers may be antigens, antibodies or small molecules. Drugs maybe selected that mimic the effects of the PRP. Such drugs are candidatesfor disease treatment.

It should be clear that any and or all types of human, plant and animaldisorders can or could be evaluated for potential drug discovery usingthe methodology outlined above. Also, as time passes new means ofevaluating a cell, tissue or organism's genetic expression will bedeveloped. These new techniques could be incorporated into theevaluation and analysis of how platelet rich plasma may be used for drugdiscovery.

Methods of Use

The PRP composition may be delivered at any suitable dose. In someembodiments, the dose may be between about 1 cc and about 3 cc, betweenabout 3 cc and about 5 cc, between about 5 cc and about 10 cc, betweenabout 10 cc and about 20 cc, or more. The dose may be deliveredaccording to a medical procedure (e.g., at specific points in aprocedure) and/or according to a schedule. For example, prior to anelective cardioversion, the PRP composition may be delivered about 24hours, about 12 hours, about 6 hours, about 2 hours, and/or about 1 hourbefore the procedure begins.

In some examples, the PRP composition may be delivered to damagedconnective tissue in or around affected joints. The PRP composition maybe delivered to an individual in need thereof by injection using asyringe or catheter. The PRP composition may also be delivered via adermal patch, a spray device or in combination with an ointment, bonegraft, or drug. It may further be used as a coating on suture, stents,screws, plates, or some other implantable medical device. Finally, itmay be used in conjunction with a bioresorbable drug or device.

In a preferred embodiment, the PRP composition is incorporated intosuture material. The PRP composition may be woven into the suturematerial. Alternatively, the suture material could be incubated with PRPprior to use. Incubation times may be from a few seconds up to anyconvenient time which may be the duration of a medical procedure. ThePRP may be incubated with the suture material from a few seconds tohours before use, such as less than 1 minute, 5-10 minutes, 10 minutesto an hour, 1-3 hours, 4-12 hours, 13-24 hours, 1-3 days, or 3-31 days.

The PRP-coated suture material may be conveniently stored in anappropriate chamber. In some embodiments, the PRP-coated suture materialmay be stored frozen and/or under reduced oxygen concentration orincreased oxygen concentration. In some embodiments, the PRP alone or incombination with a fixation device such as suture, a screw, a stent,unplantable or other device may be incubated or stored under variableconditions such as low and/or high oxygen tension, low and/or high pH,low and/or high pressure, low and/or high UV or other light conditions,low and/or high temperature. That is, conditions of oxygen tension, pH,pressure, UV or other light or temperature in the chamber vary fromphysiological and/or ambient conditions.

One specific example is a PRP conditioning chamber that has an oxygensupply that varies with time. The oxygen tension is set at or above 20%,preferably 20-25% oxygen tension, for 2-10 minutes, preferably aboutfive minutes and then slowly or abruptly changed to less than 20% oxygentension, preferably 2-10% oxygen tension, most preferably about 5%oxygen tension for 2-10 minutes, preferably about five minutes. Thisrelative hypoxic challenge may alter in a positive manner the value ofthe platelet rich plasma for tissue repair. These oxygen values may varyfrom 0-100% based on the conditions desired for a specific effect onPRP.

Storage times may vary from such as less than 1 minute, 5-10 minutes, 10minutes to an hour, 1-3 hours, 4-12 hours, 13-24 hours, 1-3 days, 3-31days, or 1-12 months or 1-5 years The PRP composition alone or incombination with the fixation device may then be used clinically asappropriate. Suture and/or other devices may also be manufacturedtogether with platelet rich plasma, incorporating the platelet richplasma composition into the device.

A simple example is incubation of suture with platelet rich plasma for10-30 minutes, preferably about 15 minutes in a chamber with 20-30%oxygen, preferably about 22% oxygen. The suture may then be used torepair an Achilles tendon or heart valve.

In alternate embodiments, a platelet rich plasma composition isincorporated into the device such as a suture, stents, screws, plates,or some other implantable medical device, during the manufacture of thedevice. The device in which platelet rich plasma is already incorporatedis then used for tissue repair.

In another embodiment, a platelet rich plasma composition is preparedand combined with a stent in an appropriate low oxygen chamber for 1-30minutes, preferably about 10 minutes. The chamber is then exposed toultraviolet light for a brief period of time, such as 1-60 seconds, 1-5minutes, or 5-15 minutes. The stent is then removed from the chamber andimplanted into a patient. It is expected that this chamber will improvethe biologic activity of the platelet rich plasma and or device.

The site of delivery of the PRP composition is typically at or near thesite of tissue damage. The site of tissue damage is determined bywell-established methods including imaging studies and patient feedbackor a combination thereof. The preferred imaging study used may bedetermined based on the tissue type. Commonly used imaging methodsinclude, but are not limited to MRI, X-ray, CT scan, Positron Emissiontomography (PET), Single Photon Emission Computed Tomography (SPECT),Electrical Impedance Tomography (EIT), Electrical Source Imaging (ESI),Magnetic Source Imaging (MSI), laser optical imaging and ultrasoundtechniques. The patient may also assist in locating the site of tissueinjury or damage by pointing out areas of particular pain and/ordiscomfort.

In some examples, a PRP composition may be used to treat a patientdiagnosed with an acute myocardial infarction. Treatment with the PRPcomposition may occur in the field or in the emergency room setting.Criteria for PRP composition treatment may include positive cardiacmarkers, ST-elevations, or new wall motion abnormalities identified onechocardiogram, for example. The decision to treat with a PRPcomposition, and the treatment location(s), may depend upon one or morecharacteristics of the myocardial infarction. For example, a myocardialinfarction may be characterized as a ST-elevation myocardial infarction(STEMI) or non-ST-elevation myocardial infarction (NSTEMI), a Q-wave ornon-Q-wave myocardial infarction, and whether they are subendocardial ortransmural. Myocardial infarctions may also be characterizedanatomically by cardiac wall region and/or the suspected blockage sitein the cardiac vasculature. Myocardial infarctions may also becharacterized as anterior, lateral, inferior, posterior, septal, orright-ventricular in location, and may involve disease or blockage ofthe left-anterior descending, left circumflex, left main,posterior-descending and right coronary arteries, for example.

In other examples, timing of the PRP preparation and application may bebased upon other treatments that are indicated in a patient with amyocardial infarction. In some instances, a PRP composition may beprepared and delivered before, during, and/or after reperfusion therapyis performed to treat an acute myocardial infarction or a previousmyocardial infarction. Reperfusion therapies may include thrombolytictherapy (such as heparin, TPA and or other pharmacologic agents),angioplasty, stenting (including bare metal stents and drug-elutingstents) or coronary artery bypass graft (CABG) surgery. In someinstances, reperfusion therapy may be associated with an increased riskof an arrhythmia, including sudden death. Also, it is believed that theetiology of reperfusion arrhythmias or reperfusion arrhythmia risk maybe different from the arrhythmia etiologies associated with themyocardial infarction itself. For example, some reperfusion arrhythmiasmay be caused by triggered activity and/or re-entry. A PRP compositionmay be prepared before or at the start of a reperfusion procedure, butnot used unless an arrhythmia occurs during the procedure. In otherreperfusion procedures, the patient may be prophylactically pre-treatedwith a PRP composition before reperfusion occurs, e.g., before guidewirepassage across an occlusion, stent positioning, stent expansion, orreestablishment of coronary flow through a bypass segment.

Results obtained by preparation of PRP from a patient exposed to athrombolytic agent is significantly different from results obtained ifthe patient has not been exposed to a thrombolytic agent systemically.Thrombolytic agents include heparin, TPA, plavix, and aspirin.

PRP was prepared prior to heparinization in a porcine model and thenafter heparinization. There were clear and significant differences asshown in the Table below. Prior to being given heparin systemically, PRPwas prepared in a standard fashion and the platelet concentration wasfound to be 5.12 times baseline. After exposure to heparin using theexact same preparation methods, the platelet concentration was found tobe 0.71 times baseline. (See Table below) This change in plateletconcentration may result in profound changes in the efficacy of the PRPas a regenerative treatment. Thus, there are clear differences in thePRPcomposition before and after exposure to heparin.

TABLE PRP Preparation Whole Blood PRP Platelet Count Platelet CountRatio Before Heparin 116 594 5.12x baseline After Heparin 685 489 0.71xbaseline

In some procedures, a PRP composition may be used as a non-specific MItreatment. Thus, the specific type and/or location of the infarction mayor may not be identified prior to delivery of the PRP composition to thepatient. For example, it may be enough to simply determine that thepatient has suffered from, or is currently suffering from, an acute MI.Thus, an ECG is not always required in order to deliver the PRP. Ofcourse, use of an ECG may be beneficial in certain circumstances. Forexample, the amount of PRP composition prepared and used may vary, basedupon whether the MI is accompanied with an elevated ST segment. In somevariations, whole blood is withdrawn prior to recording an ECG to beginpreparation of the PRP composition. The ECG may then be used todetermine an appropriate delivery mechanism simultaneously with thepreparation of the PRP composition.

In some variations, the PRP composition is injected into a heart afterthe location, type, and severity of the MI (or some fraction thereof)has been identified. In certain instances, it may be helpful to identifyone or more discrete locations within the heart to deliver the PRPcomposition in order to increase the likelihood that the treatment willbe effective.

The location of the MI may be determined or approximated using varioustechniques. For example, in some variations, diagnostic procedures suchas an electrophysiology study or an electrical mapping study of theheart may be used. In other variations, one or more imaging technologiessuch as MRI, X-ray, CT scan, Positron Emission tomography (PET), SinglePhoton Emission Computed Tomography (SPECT), Electrical ImpedanceTomography (EIT), Electrical Source Imaging (ESI), Magnetic SourceImaging (MSI), laser optical imaging and ultrasound techniques may beused. Other technologies and approaches that may be used include visualinspection during open chest surgical procedures, localized blood flowdeterminations, local electrical and structural activity, nuclearcardiology, echocardiography, echocardiographic stress test, coronaryangiography, magnetic resonance imaging (MRI), computerized tomography(CT) scans, and ventriculography.

PRP compositions that are formulated as gels or other viscous fluids maybe difficult to deliver via a needle or syringe. Thus, in variationswhere the use of a needle or syringe is desirable, it may be desirableto add a gelling and/or hardening agent to the PRP composition in situ.One or more needles or catheters may be configured to deliver the PRPcomposition and/or the agent simultaneously, or substantiallysimultaneously, to the cardiac tissue. For example, if a needle is usedto deliver the PRP composition, the needle may comprise a plurality oflumens through which the PRP composition and the agent separatelytravel. Alternatively or additionally, separate needles may be used todeliver the components to the tissue at the same time or one after theother.

The PRP composition may be delivered minimally invasively and/orsurgically. For example, the PRP composition may be delivered to theheart using a catheter inserted into the patient via the femoral vein orartery, the internal jugular vein or artery, or any other suitable veinor artery. The PRP composition may be delivered along with one or moremedical devices, instruments, or agents to treat the MI and/or othercardiac conditions.

To deliver a PRP composition to the ischemic tissue, a physician may useone of a variety of access techniques. These include surgical (e.g.,sternotomy, thoracotomy, mini-thoracotomy, sub-xiphoidal) approaches,endoscopic approaches (e.g., intercostal and transxiphoidal) andpercutaneous (e.g., transvascular, endocardial, and pericardial)approaches. Once access has been obtained, the composition may bedelivered via epicardial, endocardial, or transvascular approaches. Thecomposition may be delivered to the cardiac wall tissue or cardiacvessels in one or more locations. This includes intra-myocardial,sub-endocardial, and/or sub-epicardial administration.

Upon gaining access to the ischemic tissues of the heart, the deliverydevice may be inserted through any appropriate vessel. The distal end ofthe delivery device may be then placed against the surface of themyocardium and one or more needles may be advanced into tissue.Following delivery of one or more components of the PRP composition, theneedles, if any, may be retracted. The delivery device may then berepositioned for additional delivery of one or more components of thecomposition or may be removed from the patient. Incisions may then beclosed using standard techniques.

In practice, the beating heart may be stabilized during the delivery ofthe PRP composition. For example, in some variations, the beating heartmay be slowed or stopped by delivery of one or more drugs and/or byelectrical stimulation of the heart. For example, a heart may bestabilized using pharmacologic asystole. Alternatively or additionally,a heart may be stabilized using pacing or other algorithms that renderthe heart fairly static. These procedures may initiate various cardiacstates such as reversible initiation of asystole, fibrillation, or aprolonged refractory state. In still other embodiments, mechanicalstabilization of the cardiac tissue may be achieved using any of avariety of mechanical stabilizing systems. In some examples, acombination of stabilizing procedures may be used.

The PRP composition may be delivered during a specific portion of thecardiac cycle, and in these variations, the use of one or morestimulation electrodes to act as a pacemaker during the delivery may bedesirable. For example, the beat-to-beat period may be artificiallylengthened so as to deliver the PRP composition during a specific phaseof the cardiac cycle. In these variations, the delivery device mayinclude one or more stimulation and/or sensing electrodes. For example,sensing electrodes may be used to sense contractions of the heart,thereby allowing the delivery of composition to be timed with cardiaccontractions. It may be desirable to deliver one or more components ofthe PRP composition between contractions of the heart.

In some examples, one or more cardiac sensors may be used during thetreatment procedures. The sensors may be any suitable sensor system(e.g., an electrical sensor, a chemical sensor, a pressure sensor, anintravascular imaging sensor, or a biosensor) capable of detecting oneor more signals indicative of a cardiac contraction or heartbeat. Acardiac sensor may be used to monitor the electrical activity of theheart by picking up and amplifying electrical signals from the heart anddisplaying a visual output and/or providing an audio output. Forexample, the output may be displayed on a display interface. Thephysician may use this output to inject the needles and/or compositioninto the tissue at a specific point in the cardiac cycle. The cardiacsensor may be coupled to a cardiac stimulator to manipulate or controlthe cardiac rhythm.

In some variations, a nerve stimulator may be used to electricallymanipulate cardiac rhythm by stimulating the vagus nerve. Vagalstimulation may produce asystole (slowing or stopping of the heart).Once the vagal stimulation is stopped, the heart may return to a normalrhythm. Alternatively, the heart may be paced. Vagal stimulation, aloneor in combination with electrical pacing, may be used selectively andintermittently to allow a physician to perform delivery of one or morecomponents of the composition into a temporarily stopped heart.

Typically, vagal stimulation may slow or even prevent the heart fromcontracting. Following initial slowing or stopping of the heart, one ormore components of the PRP composition may be delivered to the heart.Following a brief interval of nerve stimulation while the delivery maybe performed, nerve stimulation may be ceased and the heart may beallowed to contract. A cardiac stimulator or pacemaker may be used tocause the heart to contract or the heart may be free to beat on its own.In some variations, one or more electrodes may be used for pacing theheart as desired. A processor may control both cardiac and nervestimulation. For example, a processor may cease nerve stimulation andautomatically begin cardiac stimulation.

The delivery system may deliver the components of the PRP composition ina prescribed ratio (e.g., a ratio of the WBCs and the platelets). Theprescribed ratio may be calculated beforehand or determined on an ad hocbasis once delivery begins. To deliver the components in the prescribedratio, the delivery device may include one or more gears having acorresponding gear ratio, one or more lumens having a proportional lumensize, or any other suitable mechanism. Some delivery devices may includeone or more mixing chambers. The multiple components may be deliveredusing separate delivery devices or may be delivered one after the otherusing the same delivery device.

The delivery devices may be advanced through a vessel adjacent to theischemic tissue to be treated. The PRP composition may be injecteddirectly into the ischemic tissue using a needle and/or a needle-tipcatheter. The PRP composition may alternatively or additionally beinfused into the vessel.

When the PRP compositions are delivered using one or more catheters, anysuitable catheter may be used. For example, the catheters may includeone or more lumens and staggered or flush tips. The catheters mayinclude needles or other devices (e.g., imaging devices) located at thedistal end, and plungers or any other control located at the proximalend. The catheters and/or other delivery devices may have differentlysized lumens to deliver multiple components of the PRP composition inthe prescribed ratio. When catheters are used, a physician may navigateto the heart using one of the routes known for accessing the heartthrough the vasculature, including but not limited to navigation to aheart chamber for epicardial, endocardial, and/or transvascular deliveryof the PRP composition.

Endocardial delivery of the PRP composition may comprise accessing atreatment site, for example, in the left ventricle of a heart, using adelivery device advanced percutaneously in an anterograde approachthrough the superior vena cava or inferior vena cava into the rightventricle. The delivery device may be passed through the interatrialseptum into the left atrium and then into the left ventricle to reachtreatment site. Alternatively, the device may be advanced using atransseptal procedure, e.g., through the intraventricular septum intothe left ventricle. In another embodiment, the PRP composition may beinjected directly into the interventricular septum from the rightventricle. An alternative endocardial delivery method may compriseaccessing the treatment site using a delivery device advancedpercutaneously in a retrograde approach through the aorta into the leftatrium and then into the left ventricle.

Transvascular delivery of compositions may comprise passing the deliverydevice through the coronary sinus into the cardiac venous system via thecardiac veins and, if needed, leaving the veins by tracking throughmyocardial tissue. An alternative transvascular delivery methodcomprises accessing a treatment site through the aorta into a coronaryartery to reach treatment site.

The devices for injecting or delivering the PRP compositions (catheteror otherwise) may include cooled parts or other temperature controlmechanisms to keep the PRP composition at a desired temperature. Variousembodiments of delivery devices may include a cooled chamber, and/or anagitator mechanism in a PRP chamber or injection chamber to preventsettling or clumping of the PRP components. For example, in somevariations, the catheter or other delivery device has a cooled lumen orlumens for keeping the PRP composition cool during delivery. Thedelivery devices may additionally or alternatively include a mixingchamber for mixing the PRP composition prior to delivery. The PRPcomposition may also be stored in an agitating/vibrating chamber, or thephysician may agitate the PRP composition once inside the deliverydevice by tilting or otherwise manipulating the device.

A practitioner may make multiple deliveries into various locations usinga single device, make multiple deliveries into various locations usingmultiple devices, make a single delivery to a single location using asingle device, or make a single delivery to a single location usingmultiple devices. The deliver devices may include at least one reusableneedle or catheter. Some embodiments may include delivery devices havingan automated dosing system (e.g., a syringe advancing system). Theautomated dosing system may allow each dose to be pre-determined anddialed in (may be variable or fixed). In some embodiments, aniontophoresis device may be used to deliver the PRP composition into theischemic tissue.

The PRP composition may alternatively or additionally be coated on oneor more devices such as, for example, sutures, stents, screws, and/orplates. Anti-arrhythmia devices, such as pacemaker leads and automaticdefibrillators may also be coated, sprayed, or dipped into the PRPcomposition prior to, simultaneously with, or subsequently toimplantation.

It may be desirable to deliver the PRP composition to the ischemictissues while avoiding coincidental delivery to other cardiac tissues orother locations adjacent to the heart. For example, the PRP compositionmay gel or harden upon delivery to prevent migration. In somevariations, a balloon catheter may be placed in the coronary sinus andinflated during delivery until the PRP composition has solidified or atleast partially immobilized. Other variations may include a pressurecontrol system on the delivery device to prevent pressure-drivenmigration of the PRP composition. Backbleed may also be prevented bykeeping the needle in place for several seconds (e.g., about 5 to about30 seconds, or about 5 to about 120 seconds) following an injection.

Sensors may be used to direct the delivery device to a desired locationand/or to deliver the PRP composition. For example, real-time recordingof electrical activity (e.g., an ECG), pH, oxygenation, metabolites suchas lactic acid, CO₂, or the like may be used. The sensors may be one ormore electrical sensors, fiber optic sensors, chemical sensors, imagingsensors, structural sensors, and/or proximity sensors that measureconductance. The sensors may be incorporated into the delivery device orbe separate from the delivery device. In some embodiments, the sensorsmay sense and/or monitor such things as needle insertion depth, bloodgas, blood pressure or flow, hemocrit, light, temperature, vibration,voltage, electric current, power, and/or impedance. The sensors mayinclude one or more imaging systems and may be coupled to anyappropriate output device, for example, a LCD or CRT monitor whichreceives and displays information.

The total volume of the PRP composition delivered to the patient may bebased on the size of the heart, the amount of the affected ischemictissue, and/or the desired outcome of the procedure. For example, thetotal volume of composition injected may be less than 15000 μL.

The number of delivery sites in the heart may be based on the type andlocation of the infarct(s), the desired location of the PRP composition,and the distance separating the desired locations. The number ofdelivery sites may range from about 1 to about 25 sites. The distanceseparating delivery sites may vary based on the desired volume ofplatelet gel to be delivered per delivery site, the desired total volumeto be delivered, and/or the condition of the ischemic tissue. At thedelivery site, the PRP composition may be injected, infused, orotherwise disposed at or adjacent to the ischemic tissue. The PRPcomposition may also be infused into the vasculature (i.e., vessels)upstream of the target site, so that it will flow towards the affectedischemic tissue.

The location of the delivery sites may vary based on the size and shapeof the ischemic tissue, and the desired extent of the treatment of thetissue. For example, the PRP composition may be delivered into theischemic tissue, and/or into the tissue that bordering the ischemictissue. Similarly, the composition may be delivered to any combinationof the regions of ischemic tissue and other cardiac tissue.

The timing of PRP delivery relative to an acute MI may be based on theseverity of the infarction, the extent of the ischemic tissue, thecondition of the patient, and the progression of any concurrent MI orarrhythmia treatments. The PRP composition may be delivered at anysuitable time. For example, it may be delivered immediately after theonset of an MI, within one hour of an MI, one to eight hours followingan MI, or three to four days after an MI after clinical stabilization ofthe patient when it is safer for the patient to undergo a separateprocedure. The timing may be based upon the level of caspase-3 in theblood. In some variations, the composition is delivered about one week,about 1 to about 3 weeks, about 1 to about 6 months, or even up to ormore than about 1 year after the MI. Other times for injectingcompositions into the ischemic tissue are also contemplated, includingprior to any potential MI, and immediately upon finding an area ofischemic tissue. Of course, compositions may be injected into theischemic tissue years after an MI.

After reperfusion, the devices and methods may be used in conjunctionwith current anti-arrhythmia therapies and/or concurrently with othermedical procedures that are generally known to increase the likelihoodof an arrhythmia. For example, and as discussed generally with respectto the methods and devices, various cardiac procedures may requireslowing (bradycardia) and/or stopping (asystole) the heart for a periodof time.

As mentioned previously, a PRP composition may additionally oralternatively be used in other cardiac procedures. These cardiacprocedures may include anti-arrhythmia procedures, procedures to correctcongenital heart defects, or other pathologies. Examples of othercardiac procedures include, but are not limited to, angioplasty,coronary artery bypass, Minimally Invasive Direct Coronary Artery Bypass(MIDCAB), off-pump coronary artery bypass, Totally Endoscopic CoronaryArtery Bypass (TECAB), aortic valve repair, aortic valve replacement,mitral valve repair, mitral valve replacement, Ross procedure, Bentallprocedure, pulmonary thromboendarterectomy, transmyocardialrevascularization (TMR), valve-sparing aortic root replacement,cardiomyoplasty, Dor procedure, heart transplantation, septal myectomy,ventricular reduction, pericardiocentesis, pericardiectomy, atrialseptostomy, Blalock-Taussig shunt procedure, Fontan procedure, Norwoodprocedure, Rastelli procedure, Maze procedure (Cox maze and minimaze),and/or pacemaker insertion. The PRP composition may used to prevent anarrhythmia associated with reperfusion of the cardiac tissue during anyof the above procedures. As is known, reperfusion may cause aspontaneous arrhythmia to occur after cardiac surgery.

The PRP composition may be used alone and or in combination with othertherapies including, but not limited to, stems cells (embryonic oradult), cord blood, drugs, genetically engineered molecules, or otherbioactive substances.

Kits

Kits may include any device, component, or combination of devices and/orcomponents described herein. For example, the kits may include one ormore preparation devices, one or more delivery devices, one or morecollection devices, and/or instructions for use. The one or morepreparation devices may be for preparing PRP and may comprise acentrifuge, for example. The one or more delivery devices may beconfigured to deliver a PRP composition comprising the PRP to damagedconnective tissue or to a region of the heart to treat an MI. The one ormore collection devices may comprise one or more syringes, apheresisneedles, or other devices for collecting blood from a patient. Thepatient may be presently suffering or have suffered an MI and/orconnective tissue damage. The components of the kit may be packaged insterile containers. The kits may comprise one or more single-usecomponents. Instructions may be in written or pictograph form, or may beon recorded media including audio tape, audio CD, video tape, DVD,CD-ROM, or the like.

In addition to the foregoing uses for the compositions, methods andsystems described herein, it will be apparent to those skilled in theart that other injured tissues, in addition to injured cardiac tissueand connective tissue, would benefit from the delivery of structuralsupport materials to treat the injuries. Non-limiting examples of suchtissues include the stomach, to reduce food intake and increase satiety;the abdominal wall, to prevent and treat hernias; and the bladder toprevent or treat incontinence. Such tissues may additionally includevascular tissues.

EXAMPLES Example 1 Treatment of Myocardial Infarction with PRP in aMouse Model

PRP was prepared using a centrifuge unit such as the Biomet GPS system,the Depuy Symphony machine, the Medtronic Magellan machine, those madeby Harvest (Plymouth, Mass.), Genesis Enterprises, Sorin Medical, andRegen Lab. Approximately 55 cc of human whole blood was drawn using astandard sterile syringe, combined with 5 cc of a citrate dextrosesolution for anticoagulation, and then spun down to isolate theplatelets according to the manufacturer's protocol. These platelets werethen resuspended in approximately 3 cc of plasma. The variousconcentrations are shown in the table in FIG. 1.

The human-derived PRP composition or saline control was injectedintramuscularly into the myocardium of a murine model after permanentleft anterior descending artery ligation. This ligation resulted in amyocardial infarction or heart attack. Ejection fraction measured atseven days by MRI. As shown in FIG. 2, the ejection fraction seven daysafter the ligation, the ejection fraction of the saline control was 26%while the ejection fraction of the group treated with PRP was 36%.

Example 2 Preparation of PRP Before Administration of AnticoagulantAgent

Whole blood is drawn immediately upon presentation to the emergency roomin a patient with a suspected heart attack. Blood may be sent for labwork but also enough extra blood is drawn to prepare platelet richplasma (PRP) before the administration of heparin, TPA, plavix, aspirinand or other pharmacologic or interventions agents. The PRP is saved fordelivery after initiation of reperfusion therapy and/or delivered priorto such intervention.

The PRP can be prepared using a variety of techniques including, but notlimited to, centrifuges, gravity filtration devices, cell sorting orothers. It can be combined with stem cells, genetic engineering ormechanical devices such as permanent or bioaborbable pacemaker or stent.The PRP can be made and then stored in a frozen or lyophilized state. Ina preferred form it would be buffered to physiologic pH but it may alsobe valuable to instill PRP at either acidic or basic pH for specificclinical indications such as ablation of an abnormal conduction pathway.In yet another embodiment, the PRP could be prepared in a form that isdepleted of neutrophils or other fractions of white blood cells eitherpartially or completely.

Example 3 Use of PRP in Drug Discovery for Ischemia-Related Conditions

Platelet rich plasma is used in an ischemia reperfusion trial (such as aheart attack) in an in-vitro, animal or human model. Analysis ofexpression of tissues or cells is done at different time points.Computer analysis of microarray output is done to seek out specificupregulation or downregulation of markers of apoptosis, cell regulationor any new or existing signaling pathways. Genes upregulated ordownregulated in response to PRP are identified. Drugs affecting theseidentified genes are tested for their effects on ischemic conditions. Onthe basis of the genetic expression, drugs useful for the treatment ofischemia or reperfusion tissue issue injury such as what can occur witha heart attack, a stroke or a traumatic injury are identified.

Example 4 Use of PRP in Drug Discovery for Cancer Treatments

A treatment employing a PRP composition is used in a cancer trial in anin-vitro, animal or human model. For example, PRP could be injected intoor around a tumor. The effects of the treatment on gene expression isdetermined using microarrays. Computer analysis of microarray output isdone to seek out specific upregulation or downregulation of markers ofapoptosis, cell regulation or any new or existing signaling pathways.Based on genetic expression in successfully treated individuals, genesare identified which are upregulated or downregulated in response toeffective treatment. Drugs affecting the identified genes are used forthe treatment of cancers of any or all types including but not limitedto brain cancer, lung cancer, breast cancer, colon cancer or otherneoplastic disorders.

Example 5 Use of PRP in Drug Discovery for Connective Tissue Injury

A PRP composition is used as in examples 3 and 4. Another disease stateor disorder is identified such as a connective tissue injury includingbut not limited to tendon, ligament, cartilage, spinal disc, muscle,bone, or others. Specific protocols are developed to evaluate thegenetic expression of cells or tissue with and without the addition ofplatelet rich plasma or its derivatives in a variety of doses orformulations. This expression is then analyzed to seek out novel targetsfor drug development for such disorders.

While methods, devices, and kits have been described in some detail hereby way of illustration and example, such illustration and example may befor purposes of clarity of understanding only. It will be readilyapparent to those of ordinary skill in the art in light of the teachingsherein that certain changes and modifications may be made theretowithout departing from the spirit and scope of the appended claims.

What is claimed is:
 1. A method of treating damaged connective tissuecomprising: delivering a composition to the patient to treat the damagedconnective tissue comprising: platelets in a concentration of about500,000/μl to about 7,000,000 μl, monocytes in a concentration of about400/μl to about 3200/μl, neutrophils in a concentration of less thanabout 5000/μl, and lymphocytes in a concentration of about 2600/μl toabout 16000/μl, wherein the composition does not include an exogenousactivator.
 2. The method of claim 1, further comprising preparing thecomposition from the whole blood of the patient.
 3. A method ofpreparing platelet-rich plasma (PRP) from a patient comprising:withdrawing blood from the patient; preparing PRP from the blood, andadministering reperfusion therapy to the patient, wherein a thrombolyticagent has not been administered to the patient prior to withdrawing theblood, and wherein the PRP comprises: platelets in a concentration ofabout 500,000/μl to about 7,000,000 μl, monocytes in a concentration ofabout 400/μl to about 3200/μl, neutrophils in a concentration of lessthan about 5000/μl, and lymphocytes in a concentration of about 2600/μlto about 16000/μl.
 4. The method of claim 3, wherein the thrombolyticagent is selected from the group consisting of heparin, tPA, PLAVIX®,and aspirin.
 5. The method of claim 3, wherein the patient has notreceived a thrombolytic agent for at least 2 hours, preferably 1 day,more preferably 2 weeks, and yet more preferably 1 month prior towithdrawing the blood.
 6. The method of claim 3, wherein preparing PRPfrom the blood comprises the steps of: obtaining a plasma fraction fromthe blood; isolating platelets from the plasma fraction; resuspendingthe platelets in a reduced amount of plasma; and adjusting the pH toprovide a pH of 7.3 to 7.5 for the resuspended platelets to provide aplatelet-rich plasma composition, wherein an activator of theplatelet-rich plasma is not added to the platelet-rich plasmacomposition.
 7. A method of identifying drug candidates for treating adisease or condition comprising administering a platelet rich plasmacomposition to an individual suffering from a disease or condition or adisease model of that disease selected from the group consisting of acell culture, computer model and animal model; monitoring the efficacyof the treatment in the individual suffering from the disease orcondition or in the model of that disease; selecting for individual orresults from the model responsive to the treatment; obtaining a blood,saliva or tissue sample from the responsive individuals or responsivemodels; analyzing the samples for a predetermined marker(s); comparingthe analysis of the samples to individuals or models of the disease notresponsive to the treatment; and identifying analytes which areresponsive to the treatment; and screening for drugs affecting theanalytes, thereby identifying drugs candidates for treating the diseaseor condition.
 8. The method of claim 7, wherein the individuals ormodels not responsive to the treatment do not have the disease orcondition.
 9. The method of claim 7, wherein the individuals or modelsnot responsive to the treatment have the disease or condition but arenot responsive to the treatment.
 10. The method of claim 7, wherein thedisease or condition is selected from the group consisting of ischemia,cancer, a disease of the immune system, a connective tissue injury, askin disease, and a disease of the nervous system.
 11. The method ofclaim 10 wherein the disease or condition is ischemia and wherein theischemia is cardiac ischemia.
 12. The method of claim 10, wherein thedisease or condition is cancer and the cancer is brain cancer.
 13. Themethod of claim 10, wherein the disease or condition is a connectivetissue injury which is a tendinosis of elbow, shoulder, or knee.
 14. Themethod of claim 10, wherein the disease or condition is a disease of thenervous system which is Parkinsons' disease.
 15. The method of claim 7,wherein the treatment comprises administering a PRP composition to atumor, cardiac tissue, or connective tissue.
 16. The method of claim 7,wherein the PRP composition comprises neutrophils at a level of 0 to 0.9of the concentration of whole blood.
 17. The method of claim 7, whereinthe analysis comprises determining a genetic profile.
 18. The method ofclaim 7, wherein the analysis comprises measuring levels of proteins,lipids, antibodies, antigens, enzymes or small molecules.
 19. A methodof disease diagnosis for a patient comprising: obtaining a blood samplefrom the patient; obtaining PRP from the blood sample; obtaining ananalysis from the PRP; and comparing the analysis of the PRP from thepatient with an analysis obtained from PRP of a population having thedisease; and determining that analysis of the PRP of the patient matchesthe analysis of the PRP of the disease population, thereby diagnosingthe disease, wherein the analysis is an immunoassay or genetic profile.20. A device comprising a platelet rich plasma composition comprising:platelets in a concentration of about 500,000/μl to about 7,000,000/μl,monocytes in a concentration of about 400/μl to about 3200/μl,neutrophils in a concentration of less than about 5000/μl, andlymphocytes in a concentration of about 2600/μl to about 16000/μl,wherein the composition does not include an exogenous activatoroptionally in combination with a fixation device selected from the groupconsisting of a stent, suture, screw and implantable device.
 21. Thedevice according to claim 20, wherein the device is a chamber andwherein conditions in the chamber include one or more of the following:low oxygen tension, high oxygen tension, low pH, high pH, low pressure,high pressure, low UV, high UV, low temperature and high temperature.